Insulin resistance results in a dysregulation of energy metabolism. The prevalence of insulin resistance is increasing at an alarming rate in the Unite States and it will continue to rise in tandem with the increase in obesity. A better understanding of insulin action at a molecular level is required to identify molecular targets for the development of therapeutics for the treatment of insulin resistance. Insulin increases glucose uptake by recruiting the GLUT4 glucose transporter from intracellular storage compartments to the plasma membrane of fat and muscle cells. It is important to understand both the mechanism responsible for the basal intracellular retention and the mechanism for the insulin signaling that induces GLUT4 translocation to the plasma .membrane. A considerable amount is known about GLUT4 trafficking and its intersection with insulin signaling, although there is still much to be learned. In this renewal application I propose a work plan that will provide novel information on three areas of GLUT4 biology. In aim 1 we will analyze the behaviors of GLUT4 mutated in the cytoplasmic FQQI, LL-based and EVEY trafficking motifs. The analysis will include detailed kinetic studies of exocytosis and intracellular trafficking of the mutants as well as studies to define the impact of the mutants on the intracellular trafficking itinerary of GLUT4. These results will provide novel information on GLUT4 sorting at a molecular level. In aim 2 we will determine the roles of rab proteins in the specialized intracellular trafficking of GLUT4. Rab proteins will be knocked-down with siRNA methods and the behavior of GLUT4 analyzed. These studies will focus on Rab10 and Rab14, two targets of the insulin-regulated AS160 rab GAP. Rab31, which has a role in basal state intracellular GLUT4 traffic, will also be studied. These results will provide novel information on the roles of rab proteins in insulin regulation of GLUT4 trafficking. In aim 3 we will characterize the behaviors of Akt-1 and Akt-2 chimeras to identify the domains responsible for the isoform-specific activity of AKT-2 in insulin signaling to GLUT4. A functional assay based on rescue of insulin-stimulated GLUT4 translocation in AKT-2 knockdown adipocytes and a total internal reflection fluorescence microscopy assay, to measure recruitment to the plasma membrane, will be used in these studies. These results will provide novel information on Akt isoform specificity in signaling to GLUT4. The proposed studies are based on our accomplishments during the past funding period. Project Narrative: Insulin stimulates glucose transport into fat and muscle cells by causing vesicles containing a protein that transports glucose (called GLUT4) to move to and fuse with the plasma membrane, a process known as translocation. The translocation of GLUT4 is defective in insulin resistance and type 2 diabetes mellitus. The objective of this research project is to study the translocation process in molecular detail, which may lead to a better understanding of defects that give rise to insulin resistance and type 2 diabetes mellitus.